Body fluid sampling container

Abstract

To provide a body fluid sampling container with which, even if body fluid is sampled so as to identify types of bacteria in leukocyte-containing fluid collected from a subject, the bacteria in the body fluid do not die, and a sufficient number of bacteria can be cultured to identify the type thereof.SOLUTION: The body fluid sampling container of the present invention is a container for sampling body fluids so as to identify types of bacteria contained in leukocyte-containing fluid collected from a subject and a non-bactericidal leukocyte function inhibitor is used.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a container for sampling body fluids, and particularly to a container for sampling leukocyte-containing body fluids collected from a subject.

Background Art

[0002] In our lives, dairy products such as milk, goat milk, or processed products thereof such as butter, yogurt, and cheese are consumed daily, and the development of the dairy industry is still highly regarded at present.

[0003] On the other hand, it is also known that the dairy industry has a large economic burden. For example, when cows are infected with bacteria and cannot ship milk, or when cows die due to the worsening of infectious diseases, the economic burden on dairy farmers is large, and the operation of dairy farming may become impossible. Therefore, appropriate measures against bacterial infections in cows and goats have become an inevitable problem for dairy farmers.

[0004] As an infectious disease, for example, mastitis is known. Mastitis is an infectious disease caused by the invasion of pathogenic microorganisms such as bacteria into the mammary glands of cows and goats and the growth of bacteria. Since milk squeezed from cows suffering from mastitis is often of low quality, it cannot be shipped to the market, making it difficult to increase profits. In addition, mastitis may recur even after being cured once, and appropriate treatment is required each time. Therefore, mastitis is known as a major problem that troubles dairy farmers.

[0005] A common treatment method for cows suffering from mastitis involves culturing bacteria in the milk to identify the type of bacteria and then administering antibacterial drugs appropriate to that type. However, the conventional method of simply sampling milk and culturing the bacteria in the milk sometimes fails to identify the bacteria, resulting in cases where drugs are administered without knowing the type of bacteria. Consequently, the cows' symptoms may not improve despite treatment, and there is a risk of them developing drug resistance to the bacteria. In light of these circumstances, there is a strong desire for the development of a more reliable method for identifying the types of bacteria in cow's milk or goat's milk, and research is actively underway to achieve this.

[0006] For example, Non-Patent Document 1 discloses the change in the number of viable pathogens during storage of milk collected from cows with subclinical mastitis, the relationship between the rate of decrease in viable bacteria during storage and the number of somatic cells, and the relationship between the rate of decrease in viable bacteria during storage and the values ​​of antimicrobial peptides, lactoferrin, and lactoperoxidase. Non-Patent Document 1 suggests that the number of viable pathogens in milk decreases while it is stored at room temperature after collection, and it is presumed that this decrease is due to leukocytes or antimicrobial components present in the milk.

[0007] Furthermore, Non-Patent Document 2 discloses further investigations into factors that reduce the number of viable pathogens during the storage of milk collected from cows with subclinical mastitis. Non-Patent Document 2 suggests that most pathogens contained in milk with a high somatic cell count are reduced during storage at 15-25°C by both cellular components (including leukocytes) and antimicrobial components contained in the milk, and it is disclosed that cellular components, in particular, significantly reduced the number of bacteria. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Hisaeda K, Koshiishi T, Watanabe M,Miyake H, Yoshimura Y, Isobe N (2016) Change in viable bacterial count during preservation of milk derived from dairy cows with subclinical mastitis and its relationship with antimicrobial components in milk. The Journal of Veterinary Medical Science 78(8):1245-1250. [Non-Patent Document 2] Koshiishi T, Watanabe M, Miyake H, Hisaeda K, Isobe N (2017) Cellular and soluble components counts decrease the viable pathogen in milk from dairy cows with subclinical mastitis. The Journal of Veterinary Medical Science 79(8):1389-1393. [Overview of the project] [Problems that the invention aims to solve]

[0009] From a dairy farming perspective, there is a strong need to more reliably identify the type of bacteria in order to provide appropriate treatment to cattle and other animals infected with bacteria.

[0010] On the other hand, as disclosed in Non-Patent Documents 1 and 2, it is known that the number of bacteria in milk decreases over time due to the antibacterial action of leukocytes contained in milk. Therefore, even if the bacteria in the milk are cultured after being collected in a sampling container, the bacteria die between the time the milk is collected and the start of culture, making it impossible to culture the bacteria and identify the type of bacteria.

[0011] The present invention has been made in view of the above problems, and its object is to provide a body fluid sampling container that allows for the cultivation of a sufficient number of bacteria to identify the type of bacteria in a leukocyte-containing body fluid taken from a subject, without the bacteria in the body fluid being killed. [Means for solving the problem]

[0012] To achieve the above objective, the inventors, through diligent research, discovered that by adding a leukocyte function inhibitor that does not have bactericidal properties to a leukocyte-containing body fluid collected from a subject, the bacteria are not killed even when leukocytes and bacteria coexist, and a sufficient number of bacteria contained in the leukocyte-containing body fluid can be cultured to identify their species. This led to the completion of the present invention.

[0013] Specifically, the bodily fluid sampling container according to the present invention is a container for sampling bodily fluids containing leukocytes collected from a subject in order to identify the types of bacteria contained in said bodily fluids, and is characterized by containing a leukocyte function inhibitor that does not have bactericidal properties.

[0014] According to the bodily fluid sampling container of the present invention, even when leukocyte-containing bodily fluid is collected and stored in the container from a subject infected with bacteria, the bacteria do not die within the container because it contains a leukocyte function inhibitor that does not have bactericidal properties, and a sufficient number of bacteria can be cultured to identify their type. In other words, compared to when bacteria in leukocyte-containing bodily fluid are cultured in a conventional sampling container, this bodily fluid sampling container can suppress the antibacterial action of leukocytes, and since the leukocyte function inhibitor itself does not risk killing bacteria, bacteria contained in leukocyte-containing bodily fluid can be cultured more reliably, and the type of bacteria can be identified more reliably. Therefore, appropriate treatment can be given to the subject after considering the type of bacteria identified. In addition, by pre-packaging the container with a leukocyte function inhibitor that does not have bactericidal properties, it becomes easy to carry, and leukocyte-containing bodily fluid collected from the subject can be stored immediately.

[0015] In the bodily fluid sampling container according to the present invention, the bodily fluid sampling container may include filter paper inside the container, and the filter paper may contain the non-bactericidal leukocyte function inhibitor.

[0016] According to the bodily fluid sampling container of the present invention, after collecting leukocyte-containing bodily fluid from a subject into the container, if the presence of filter paper can be visually confirmed within the container, it can be determined that non-bactericidal leukocyte function inhibitors have not dispersed outside the container. This allows for accurate determination of whether or not non-bactericidal leukocyte function inhibitors have dispersed outside the container even when leukocyte-containing bodily fluid is collected directly and forcefully from the subject into the container, at a stage after the bodily fluid collection. Therefore, compared to bodily fluid sampling containers that do not use filter paper, bacteria contained in leukocyte-containing bodily fluid can be cultured more reliably, and the type of bacteria can be identified more reliably. Furthermore, this bodily fluid sampling container can accurately make the above determination even when the leukocyte-containing bodily fluid has color.

[0017] In the bodily fluid sampling container according to the present invention, the non-bactericidal leukocyte function inhibitor can be colchicine or EDTA (ethylenediaminetetraacetic acid).

[0018] In this case, the colchicine content can be 0.1 μg / mL to 1 μg / mL based on the volume of the leukocyte-containing body fluid, and the EDTA content can be 0.2 mg / mL to 5 mg / mL based on the volume of the leukocyte-containing body fluid.

[0019] In the bodily fluid sampling container according to the present invention, by using colchicine or EDTA (ethylenediaminetetraacetic acid) as a leukocyte function inhibitor that does not have bactericidal properties, the antibacterial action of leukocytes can be suppressed, and since these drugs themselves do not pose a risk of killing bacteria, the bodily fluid sampling container can be made capable of culturing a sufficient number of bacteria for identification of their species.

[0020] In the body fluid sampling container according to the present invention, the leukocyte-containing body fluid can be selected from the group consisting of saliva, milk, cerebrospinal fluid, sputum, and urine.

[0021] In the body fluid sampling container according to the present invention, the subject can be selected from the group consisting of humans, cows, goats, pigs, dogs, cats, and chickens.

[0022] In the body fluid sampling container according to the present invention, the subject is a cow, and the leukocyte-containing body fluid is milk collected from a cow suffering from mastitis.

[0023] According to the body fluid sampling container of the present invention, even if milk collected from a cow suffering from mastitis is collected and stored, bacteria do not die in the container, and a sufficient number of bacteria can be cultured to identify their types. Therefore, the types of bacteria contained in the milk can be identified. Thereby, the bacteria causing mastitis can be identified, and appropriate treatment can be administered to the cow.

[0024] In the body fluid sampling container according to the present invention, the subject is a goat, and the leukocyte-containing body fluid is milk collected from a goat suffering from mastitis.

[0025] According to the body fluid sampling container of the present invention, even if milk collected from a goat suffering from mastitis is collected and stored, bacteria do not die in the container, and a sufficient number of bacteria can be cultured to identify their types. Therefore, the types of bacteria contained in the goat's milk can be identified. Thereby, the bacteria causing mastitis can be identified, and appropriate treatment can be administered to the goat.

Advantages of the Invention

[0026] The bodily fluid sampling container according to the present invention allows for the cultivation of a sufficient number of bacteria in the leukocyte-containing bodily fluid collected from a subject to identify the type of bacteria. Specifically, compared to culturing bacteria in leukocyte-containing bodily fluids using conventional sampling containers, this bodily fluid sampling container can suppress the antibacterial action of leukocytes, and the leukocyte function inhibitor itself does not risk killing bacteria. Therefore, a sufficient number of bacteria in the leukocyte-containing bodily fluid can be cultivated to identify the type of bacteria, enabling more reliable identification of bacterial species. As a result, appropriate treatment can be administered to the subject after considering the type of bacteria identified. Furthermore, by pre-packaging the container with a leukocyte function inhibitor that does not have bactericidal properties, it becomes easy to transport, and the leukocyte-containing bodily fluid collected from the subject can be stored immediately. [Brief explanation of the drawing]

[0027] [Figure 1] This is a front view of a body fluid sampling container according to this embodiment. [Figure 2] This is a front view of a body fluid sampling container equipped with filter paper according to this embodiment. [Figure 3] This is an explanatory diagram showing an example of the use of a body fluid sampling container equipped with filter paper according to this embodiment. [Modes for carrying out the invention]

[0028] The following descriptions of embodiments for carrying out the present invention will be based on the drawings. The following descriptions of preferred embodiments are essentially illustrative and are not intended to limit the present invention, its application methods, or its uses.

[0029] The bodily fluid sampling containers (1, 10) according to this embodiment will be described below.

[0030] One embodiment of the present invention is a body fluid sampling container (1, 10) for sampling a body fluid (5) containing leukocytes taken from a subject in order to identify the types of bacteria contained in the body fluid, wherein the container contains a leukocyte function inhibitor (3) that does not have bactericidal properties.

[0031] As shown in Figure 1, the body fluid sampling container (1) according to this embodiment can be, for example, a container (2) coated on the inside with a leukocyte function inhibitor (3) that does not have bactericidal properties.

[0032] In this embodiment, the container (2) can be, for example, a glass container such as a glass test tube or vial, or a resin container such as a polystyrene tube or polypropylene tube. In this embodiment, although not shown, it is preferable to use these containers (2) in combination with a lid that fits the shape of the opening at the end of the container (2). The lid can seal the opening of the container (2), thereby preventing the leukocyte-containing fluid (5) or other contents inside the container (2) from splashing out of the container (2). However, the container (2) is not limited to these and includes anything that can be used as a container.

[0033] In this embodiment, the subjects are not particularly limited and include all animals, but mammals and birds are preferred because they have white blood cells. Examples of subjects include humans, cattle, goats, pigs, dogs, cats, and chickens. Furthermore, the subjects include not only healthy subjects but also subjects suffering from illness. The illness is preferably a bacterial infection. After collecting white blood cell-containing body fluid (5) from a subject suffering from a bacterial infection into a body fluid sampling container (1, 10) according to this embodiment, the type of bacteria that caused the infection can be identified by culturing the bacteria in the white blood cell-containing body fluid (5), and appropriate treatment can be given to the subject. The bacteria that caused the infection are not limited to one type, but may be two or more types.

[0034] In this specification, leukocyte-containing body fluid (5) refers to a body fluid that can be collected from a subject and contains leukocytes. For example, the leukocyte-containing body fluid (5) can be the subject's saliva, milk, cerebrospinal fluid, sputum, or urine. Leukocytes usually refer to leukocytes contained in the subject, but may also be leukocytes introduced from an external source (i.e., leukocytes not originating from the subject). In other words, in this embodiment, a leukocyte-containing body fluid (5) containing at least one of leukocytes contained in the subject and leukocytes introduced from an external source can be used.

[0035] In this embodiment, the subject is preferably a cow suffering from a bacterial infection, and the leukocyte-containing body fluid (5) is preferably milk. After collecting and storing milk in the body fluid sampling containers (1, 10) according to this embodiment, the bacteria in the milk can be cultured to identify the type of bacteria that caused the infection. Bacterial infections include, but are not limited to, bacterial infections such as mastitis, respiratory disease syndrome, and bovine E. coli infection, and include all known bacterial infections in cattle.

[0036] In this embodiment, the subject is preferably a goat suffering from a bacterial infection, and the leukocyte-containing body fluid (5) is preferably goat's milk. After collecting and storing goat's milk in the body fluid sampling containers (1, 10) according to this embodiment, the bacteria in the goat's milk can be cultured to identify the type of bacteria that caused the infection. The bacterial infection includes, but is not limited to, bacterial infections such as mastitis, and includes all known bacterial infections in goats.

[0037] In this specification, a non-bactericidal leukocyte function inhibitor (3) refers to a drug that suppresses the antibacterial action of leukocytes without killing bacteria. In this embodiment, the non-bactericidal leukocyte function inhibitor (3) can be, for example, colchicine or EDTA (ethylenediaminetetraacetic acid). Colchicine and EDTA are preferred as non-bactericidal leukocyte function inhibitors (3) because they can suppress the antibacterial action of leukocytes and these drugs themselves do not pose a risk of killing bacteria. However, the non-bactericidal leukocyte function inhibitor (3) is not limited to these drugs and includes all drugs known to those skilled in the art as drugs that suppress the antibacterial action of leukocytes without killing bacteria.

[0038] In the body fluid sampling containers (1, 10) according to this embodiment, the colchicine content can be selected within the range of 0.02 μg / mL to 2.5 μg / mL, preferably within the range of 0.1 μg / mL to 2.5 μg / mL, based on the volume of the leukocyte-containing body fluid (5). If the colchicine content is less than 0.02 μg / mL, the antibacterial action of leukocytes cannot be suppressed, and bacteria in the leukocyte-containing body fluid (5) may be killed. Furthermore, it is preferable that the colchicine content be 0.1 μg / mL or more. This is preferable because it can sufficiently suppress the antibacterial action of leukocytes. On the other hand, there is no particular upper limit set for the colchicine content, but even if the colchicine content exceeds 2.5 μg / mL, there is no significant change in the ability to suppress the antibacterial action of leukocytes, so the upper limit for the colchicine content is set at 2.5 μg / mL. Furthermore, the colchicine content is more preferably 0.1 μg / mL to 1.0 μg / mL based on the volume of the leukocyte-containing body fluid (5). In this case, the antibacterial action of leukocytes can be sufficiently suppressed, so that bacteria in the leukocyte-containing body fluid (5) do not die in the body fluid sampling container (1, 10) according to this embodiment, and a sufficient number of bacteria can be cultured to identify their type.

[0039] In the body fluid sampling containers (1, 10) according to this embodiment, the EDTA content can be selected within the range of 0.2 mg / mL to 5 mg / mL based on the volume of the leukocyte-containing body fluid (5). If the EDTA content is less than 0.2 mg / mL, the antibacterial action of leukocytes cannot be suppressed, and there is a risk that the bacteria in the leukocyte-containing body fluid (5) will be killed. On the other hand, there is no particular upper limit set for the EDTA content, but even if the EDTA content exceeds 5 mg / mL, there is no significant change in the ability to suppress the antibacterial action of leukocytes, so the upper limit for the EDTA content is set at 5 mg / mL. Furthermore, it is more preferable that the EDTA content be between 0.2 mg / mL and 1 mg / mL based on the volume of the leukocyte-containing body fluid (5). In this case, the antibacterial action of leukocytes can be sufficiently suppressed, so that the bacteria in the leukocyte-containing body fluid (5) in the body fluid sampling containers (1, 10) according to this embodiment are not killed, and a sufficient number of bacteria can be cultured to identify their type.

[0040] The body fluid sampling containers (1, 10) according to this embodiment may optionally contain additives other than the leukocyte function inhibitor (3) that does not have bactericidal properties.

[0041] As shown in Figure 2, the body fluid sampling container (10) according to this embodiment can also be a container (2) equipped with, for example, a filter paper (4) to which a non-bactericidal leukocyte function inhibitor (3) has been added.

[0042] In this embodiment, the container (2), the non-bactericidal leukocyte function inhibitor (3), and additives can be the same as those described above. The subject and the leukocyte-containing body fluid (5) can also be the same as those described above.

[0043] In this embodiment, a non-bactericidal leukocyte function inhibitor (3) is added to the filter paper (4) in advance, and this is used as a body fluid sampling container (10). With such a body fluid sampling container (10), after collecting leukocyte-containing body fluid (5) from the subject into the container, if the presence of the filter paper (4) can be visually confirmed inside the container (2), it can be determined that the non-bactericidal leukocyte function inhibitor (3) has not been scattered outside the container (2).

[0044] In dairy farming, it is considered preferable to directly milk cows and other animals into a body fluid sampling container. However, because the force of this milking is quite strong, there is a problem that when the milk enters the body fluid sampling container (1) with force, the coating of the non-bactericidal leukocyte function inhibitor (3) inside the container peels off and scatters outside the container (2). Also, since milk is often white, even if the non-bactericidal leukocyte function inhibitor (3) has scattered outside the container (2), the person collecting the sample may not notice. On the other hand, as described above, with the body fluid sampling container (10) according to this embodiment, it is possible to accurately determine whether or not the non-bactericidal leukocyte function inhibitor (3) has scattered outside the container (2) simply by checking whether or not filter paper (4) is present inside the container (2) after collecting the leukocyte-containing body fluid (5). Furthermore, the body fluid sampling container (10) according to this embodiment is suitable because it can accurately make the above determination even when the leukocyte-containing body fluid (5) has color. Therefore, the bodily fluid sampling container (10) according to this embodiment is suitable for use, for example, when directly collecting and storing milk from a cow's teat.

[0045] In this embodiment, it is preferable to use a filter paper (4) whose diameter is the same as the inner diameter of the container (2) and whose shape is the same as the internal shape of the container. Such a filter paper (4) can be fixed at any position inside the container (2), making it easier to add a non-bactericidal leukocyte function inhibitor (3) to the filter paper (4). Any filter paper available on the market can be used as the filter paper (4).

[0046] The method for manufacturing the bodily fluid sampling containers (1, 10) according to this embodiment will be described below.

[0047] A body fluid sampling container (1) coated with a non-bactericidal leukocyte function inhibitor (3) inside a container (2), as shown in Figure 1, can be prepared, for example, by the following procedure. First, a solution is prepared by dissolving the non-bactericidal leukocyte function inhibitor (3) in ethanol. Next, an appropriate amount of the prepared solution is transferred into the container (2) and coated by air drying. Furthermore, the container (2) is placed in a clean bench and sterilized by irradiating it with an ultraviolet lamp for 10 minutes. Although not shown in the diagram, the body fluid sampling container (1) can be prepared by finally sealing the mouth of the sterilized container (2) with a lid.

[0048] In such a body fluid sampling container (1), the non-bactericidal leukocyte function inhibitor (3) adheres to the inside of the container (2), thus preventing the non-bactericidal leukocyte function inhibitor (3) from scattering inside or outside the container (2), and making it easy to carry.

[0049] In the above preparation method, ethanol is used as a solvent to dissolve the non-bactericidal leukocyte function inhibitor (3). In this embodiment, ethanol is suitable as a solvent, as it dissolves the non-bactericidal leukocyte function inhibitor (3) more easily than when water is used, and it dries quickly after being transferred to the container (2), making it very convenient. However, the solvent is not limited to ethanol and includes any solvent that can be used.

[0050] In this embodiment, it is preferable to sterilize the container (2) after coating it with a non-bactericidal leukocyte function inhibitor (3) by irradiation with ultraviolet light or the like. This sterilizes any bacteria present in the container (2) and prevents the proliferation of bacteria during use. Furthermore, the method of sterilizing the container (2) is not limited to irradiation with ultraviolet light.

[0051] A body fluid sampling container (10) equipped with filter paper (4), as shown in Figure 2, can be prepared, for example, by the following procedure. The procedure described here assumes the use of a test tube as the container (2), colchicine as a non-bactericidal leukocyte function inhibitor (3), and 10 μg of colchicine in the container (2). First, the filter paper (4) is placed in the test tube. Next, 1 mg of colchicine is measured out, placed in a separate container, and 10 mL of ethanol is added and the mixture is thoroughly stirred to prepare a 100 μg / mL colchicine solution. 100 μL of this prepared 100 μg / mL colchicine solution is transferred to the filter paper (4) in the test tube and allowed to air dry, thereby adding 10 μg of colchicine to the filter paper (4). Finally, this test tube is placed in a clean bench and sterilized by irradiation with an ultraviolet lamp for 10 minutes. Although not shown in the diagram, the body fluid sampling container (10) can be prepared by finally sealing the mouth of the sterilized test tube with a lid.

[0052] The above preparation method is particularly suitable when adding a small amount of a non-bactericidal leukocyte function inhibitor (3) to a container (2). To illustrate with colchicine as an example, accurately measuring 10 μg of colchicine with a spatula and transferring it to a test tube is practically impossible because the amount of colchicine is too small. On the other hand, as mentioned above, by first preparing a solution containing colchicine separately, transferring an appropriate amount of this solution to filter paper (4), and allowing it to air dry, even a small amount of colchicine such as 10 μg can be added with near accuracy, making this method preferable.

[0053] In the above preparation method, ethanol is used as the solvent for dissolving colchicine, which is preferable for the reasons mentioned above; however, the solvent for dissolving colchicine is not limited to ethanol.

[0054] In this embodiment, it is preferable to sterilize the inside of the test tube by ultraviolet irradiation or the like after adding colchicine to the filter paper (4). This sterilizes any bacteria present in the test tube and prevents them from multiplying before use. Furthermore, the method of sterilizing the container (2) is not limited to ultraviolet irradiation.

[0055] The above preparation method describes the case where 10 μg of colchicine is added to the filter paper (4). However, the solution concentration and other conditions can be appropriately changed depending on the amount of non-bactericidal leukocyte function inhibitor (3) added. Furthermore, the above preparation method can also be used when EDTA is used as the non-bactericidal leukocyte function inhibitor (3).

[0056] An example of the use of the bodily fluid sampling containers (1, 10) according to this embodiment will be described below.

[0057] The bodily fluid sampling containers (1, 10) according to this embodiment can be used, for example, to identify the type of bacteria in leukocyte-containing bodily fluid (5) collected from a subject when the subject suffers from a bacterial infection.

[0058] In this embodiment, the type of bacteria in the leukocyte-containing body fluid (5) can be identified, for example, by following the steps (a) to (c) below: (a) The leukocyte-containing body fluid (5) of the subject is collected in a body fluid sampling container (1, 10) according to this embodiment and stored. (b) The body fluid in the body fluid sampling container (1, 10) is cultured under appropriate conditions. (c) A staining test and a biochemical characterization test are performed to identify the type of bacteria based on the morphology of the bacteria, the staining test results, and the biochemical characterization test results.

[0059] The following describes the case in which a body fluid sampling container (10) equipped with filter paper (4) is used. As shown in Figure 3, in this embodiment, in procedure (a), a leukocyte-containing body fluid (5) is collected from the subject into the body fluid sampling container (10), and the filter paper (4) containing a non-bactericidal leukocyte function inhibitor (3) is immersed in the leukocyte-containing body fluid (5). Subsequently, although not shown, the opening at the end of the container (2) is sealed with a lid, and the body fluid sampling container (10) is shaken for at least 10 seconds to thoroughly dissolve the non-bactericidal leukocyte function inhibitor (3) contained in the filter paper (4) into the leukocyte-containing body fluid (5). This suppresses the antibacterial action of the leukocytes in the leukocyte-containing body fluid (5). Note that the filter paper (4) does not need to be removed from the body fluid sampling container (10), and the leukocyte-containing body fluid (5) can be stored as is.

[0060] In procedure (a), once it is confirmed that the non-bactericidal leukocyte function inhibitor (3) has been sufficiently dissolved, the procedure may be moved immediately to procedure (b), or the leukocyte-containing body fluid (5) may be stored in the body fluid sampling container (10) for several hours or several days before proceeding to procedure (b).

[0061] In procedure (a), the method for collecting the leukocyte-containing body fluid (5) is to directly collect the leukocyte-containing body fluid (5) from the subject into the body fluid sampling container (10), or to first put the leukocyte-containing body fluid (5) into another container and then transfer it into the body fluid sampling container (10) using a pipette or the like.

[0062] In procedure (a), the time for storing the leukocyte-containing body fluid (5) in the body fluid sampling container (10) is 10 seconds or more, and is not particularly limited. As mentioned above, the body fluid sampling container (10) is shaken for at least 10 seconds to sufficiently dissolve the non-bactericidal leukocyte function inhibitor (3) in the leukocyte-containing body fluid (5), so this may be considered the 10-second storage time. On the other hand, in order to sufficiently suppress the antibacterial action of the leukocytes contained in the leukocyte-containing body fluid (5), the body fluid may be stored in the body fluid sampling container (10) for a period of 30 minutes to 100 hours after collection. Since bacteria will not be killed even if the body fluid is stored for several days or more after collection in the body fluid sampling container (10), there is no particular upper limit on the storage time.

[0063] In procedure (b), for example, the leukocyte-containing body fluid (5) stored in a body fluid sampling container (10) is seeded onto an agar plate, and the bacteria in the body fluid are cultured. Typically, culturing at 37°C for 24 hours allows for culturing a sufficient number of bacteria to identify the type of bacteria. However, the culture conditions can be changed as appropriate.

[0064] In procedure (c), staining tests and biochemical characterization tests are performed to identify the type of bacteria based on the morphology of the bacteria, the staining test results, and the biochemical characterization test results. In procedure (c), staining tests include, for example, Gram staining, capsular staining, acid-fast bacilli staining, metachromatic body staining, silver staining, Jimenez staining, India ink staining, and KOH staining. Biochemical characterization tests include, for example, glucose decomposition tests, respiratory function tests, amino acid analysis tests, enzyme and toxin production confirmation tests, cell surface structure and function tests, and pigment production tests. Based on these staining tests, biochemical characterization tests, and the results of the morphological analysis of the bacteria, the type of bacteria contained in the leukocyte-containing body fluid (5) of the subject can be identified. However, the staining tests and biochemical characterization tests are not limited to these. Any method known to those skilled in the art for identifying the type of bacteria can be used.

[0065] The types of bacteria that can be identified in this embodiment include, for example, Enterobacter sp., Streptococcus uberis, Corynebacterium epidermidicanis, Mannheimia varigena, Escherichia coli, Enterococcus faecium, Klabsiella pneumoniae, Enterococcus saccharolyticus, Corynebacterium amycolatum, Enterococcus faecalis, Stenotrophomonas maltophilia, Proteus vulgaris / penneri, Staphylococcus simulans, Streptococcus sp., Staphylococcus haemolyticus, Streptococcus agalactiae ssp dygalactiae, Staphylococcus chromogenes, and Streptococcus dysgalactiae. However, the types of bacteria that can be identified in this embodiment are not limited to these.

[0066] Another embodiment of the present invention is a kit (not shown) comprising a body fluid sampling container (1, 10) containing a non-bactericidal leukocyte function inhibitor (3).

[0067] The kit according to this embodiment includes at least a body fluid sampling container (1, 10) containing a non-bactericidal leukocyte function inhibitor (3). The kit also optionally includes instruments such as pipettes, culture equipment, staining test equipment, and biochemical property test equipment. However, it is not limited to these instruments, and includes all instruments known to those skilled in the art as necessary for identifying bacteria.

[0068] In the kit according to this embodiment, the body fluid sampling containers (1, 10) described above can be used. Therefore, in this embodiment, for example, the container (2), the non-bactericidal leukocyte function inhibitor (3), the filter paper (4), and the additives can be the same as those used in the body fluid sampling containers (1, 10) according to the above embodiment. In addition, the subject and the leukocyte-containing body fluid (5) can also be the same as those used in the body fluid sampling containers (1, 10) according to the above embodiment.

[0069] According to the kit of this embodiment, even if a leukocyte-containing body fluid (5) is collected from a bacterial-infected subject in a body fluid sampling container (1, 10) and stored, the bacteria do not die in the container because it contains a leukocyte function inhibitor (3) that does not have bactericidal properties, and a sufficient number of bacteria can be cultured to identify their type. Furthermore, since the kit includes all the necessary tools to identify the type of bacteria, dairy farmers can investigate the bacteria causing infections in their cattle or goats on their own, and provide appropriate treatment to the cattle or goats quickly. [Examples]

[0070] The following are examples illustrating the bodily fluid sampling container according to the present invention in detail.

[0071] Example 1 (Identification of bacterial species and viable cell count when milk from a cow suffering from mastitis is collected in a colchicine-containing bodily fluid sampling container or a colchicine-free bodily fluid sampling container, and bacteria in the milk are cultured.) First, test tubes containing filter paper with 1 μg and 10 μg of colchicine were prepared. A 12.7 mm diameter filter paper was placed in each of the two test tubes, 1 mg of colchicine was measured out and placed in a separate container, and 10 mL of ethanol was added and stirred well to prepare a 100 μg / mL colchicine solution. 10 μL and 100 μL of the 100 μg / mL colchicine solution prepared in this way were transferred to the filter paper in each test tube and allowed to air dry, adding 1 μg and 10 μg of colchicine to the filter paper, respectively. Next, the two test tubes were placed in a clean bench and sterilized by irradiation with an ultraviolet lamp for 10 minutes, and the mouths of the test tubes were sealed with lids to prepare test tubes with filter paper. Then, milk was collected from the udders of 28 dairy cows suffering from mastitis. Ten mL of collected milk was transferred to test tubes equipped with filter paper containing 1 μg and 10 μg of prepared colchicine, respectively. The colchicine concentrations were adjusted to 0.1 μg / mL and 1 μg / mL based on the volume of milk. After storing these milk samples in test tubes for 5 hours, they were inoculated onto agar plates and cultured at 37°C for 24 hours to allow bacteria to develop. As a control, collected milk was transferred to test tubes without added colchicine, and samples were inoculated onto agar plates immediately after preparation (0 hours of storage) or after 5 hours of storage, and cultured at 37°C for 24 hours to allow bacteria to develop. After culturing the bacteria, the number of colonies formed was counted to calculate the viable cell count. Staining and biochemical characterization tests were also performed on the cultured bacteria, and the bacterial species were identified based on the morphology, staining test results, and biochemical characterization test results. Tables 1-3 show the identified bacterial names and viable cell counts for milk samples 1-28. Furthermore, the milk sample 23 in Table 2 contained two types of bacteria, and the types of bacteria were identified.

[0072] [Table 1]

[0073] [Table 2]

[0074] [Table 3]

[0075] The results in Tables 1-3 show that the number of viable bacteria when bacteria in milk were cultured after being stored for 5 hours without colchicine was lower than the number of viable bacteria when the milk was cultured immediately after collection. This is thought to be because the antibacterial action of leukocytes contained in milk kills the bacteria. In particular, when bacteria in milk samples 7, 8, 11-13, and 22 were cultured after being stored for 5 hours without colchicine, the bacteria were completely killed, and it was not possible to identify the types of bacteria.

[0076] The results in Tables 1-3 show that the number of viable bacteria in milk cultured after 5 hours of storage with 0.1 μg / mL or 1.0 μg / mL of colchicine added tended to be higher than the number of viable bacteria in milk cultured after 5 hours of storage without colchicine addition. This is thought to be because colchicine suppresses the antibacterial action of leukocytes without killing the bacteria contained in the milk. For milk samples 7, 8, 11-13, and 22, whose bacterial species could not be identified in the above experiment, bacteria could be cultured under the condition of adding colchicine and storing the milk for 5 hours before culturing the bacteria, and the bacterial species in these samples could also be identified.

[0077] The results in Tables 1-3 show that the body fluid sampling container according to this embodiment allows colchicine to suppress the antibacterial action of leukocytes contained in milk, enabling the cultivation of a sufficient number of bacteria to identify the type of bacteria, and thus allowing for more reliable identification of bacterial species.

[0078] Example 2 (Investigation of viable bacterial counts in goat milk with or without EDTA) Lipopolysaccharide (1 μg / 5 mL physiological saline) was injected into the udder of a goat. The day after injection, goat's milk was collected and centrifuged at 5,000 g for 15 minutes. The goat's milk was adjusted so that the precipitated leukocyte content was approximately 2,000,000 to 5,000,000 cells / mL. Subsequently, bacteria (Klebsiella pneumoniae) and EDTA were added to achieve a bacterial content of 2,000 cells / mL and EDTA content of 0.2 mg / mL or 5 mg / mL, respectively, and the goat's milk was stored for 3 hours. After that, the goat's milk was seeded onto an agar plate, and the bacteria in the goat's milk were cultured at 37°C for 24 hours. The EDTA used was the same as when colchicine was added to filter paper in Example 1. As a control, goat's milk without EDTA was prepared, and samples were inoculated onto agar plates immediately after preparation (0 hours of storage) or after 3 hours of storage. The bacteria in the goat's milk were cultured at 37°C for 24 hours. After culturing the bacteria, the number of colonies formed was counted to calculate the number of viable bacteria. The results for the number of viable bacteria for goat's milk samples 1 and 2 are shown in Table 4. Note that the bacteria added were of a pre-identified species, so the bacterial species were not identified. The injection of lipopolysaccharide into the goats was performed to increase the number of white blood cells in the goats' bodies.

[0079] [Table 4]

[0080] The results in Table 4 show that when bacteria in goat's milk were cultured after being stored for 3 hours without EDTA, the number of viable bacteria was 0 in both cases, and this value was lower than when bacteria in goat's milk were cultured immediately after collection. This is thought to be because the antibacterial action of leukocytes contained in goat's milk kills the bacteria (Klebsiella pneumoniae).

[0081] On the other hand, it was found that the number of viable bacteria in goat's milk cultured after being stored for 3 hours with EDTA 0.2 mg / mL or 5.0 mg / mL added was higher than the number of viable bacteria in goat's milk cultured after being stored for 3 hours without colchicine added. This is thought to be because EDTA suppresses the antibacterial action of leukocytes without killing the bacteria contained in goat's milk.

[0082] The results in Table 4 show that EDTA-treated goat's milk also suppressed the antibacterial activity of leukocytes, allowing for the cultivation of a sufficient number of bacteria (Klebsiella pneumoniae) present in the goat's milk.

[0083] Example 3 (Investigation of viable bacterial count in bovine urine with or without colchicine) Lipopolysaccharide (1 μg / 5 mL physiological saline) was injected into the udder of a goat. The day after injection, goat's milk was collected and centrifuged at 5,000 g for 15 minutes. The precipitated leukocyte content was adjusted with bovine urine to approximately 2,000,000 to 5,000,000 cells / mL. Subsequently, bacteria (E. coli), colchicine, and bovine urine were added to prepare bovine urine samples, with a bacterial (E. coli) content of 2,000 cells / mL and colchicine content of 0.02 μg / mL, 0.1 μg / mL, 0.5 μg / mL, and 2.5 μg / mL. As in Example 1, colchicine was added to filter paper. After storing each bovine urine sample for 3 hours, the urine samples were seeded onto agar plates and the bacteria in the bovine urine were cultured at 37°C for 24 hours. As a control, bovine urine samples without colchicine were prepared and inoculated onto agar plates immediately after preparation (stored for 0 hours) or after 3 hours. The bacteria in the bovine urine were cultured at 37°C for 24 hours. After culturing the bacteria, the number of colonies formed was counted to calculate the number of viable bacteria. The results for the number of viable bacteria for bovine urine samples 1-3 are shown in Table 5. Note that since the bacteria added were of a pre-identified species, bacterial identification was not performed. Furthermore, the injection of lipopolysaccharide into the goats was performed to increase the number of white blood cells in the goats' bodies.

[0084] [Table 5]

[0085] The results in Table 5 show that the number of viable bacteria when cultured from bovine urine after being stored for 3 hours without colchicine tended to be lower than the number of viable bacteria when cultured from bovine urine immediately after collection. This is thought to be because the antibacterial action of leukocytes contained in bovine urine kills the bacteria (E. coli).

[0086] On the other hand, it was found that the number of viable bacteria when bacteria in bovine urine were cultured after being stored for 3 hours in bovine urine samples to which colchicine was added at concentrations of 0.02 μg / mL, 0.1 μg / mL, 0.5 μg / mL, and 2.5 μg / mL tended to be higher than the number of viable bacteria when bacteria in bovine urine were cultured after being stored for 3 hours in bovine urine samples without colchicine addition. This is thought to be because colchicine suppresses the antibacterial action of leukocytes without killing the bacteria (E. coli) contained in bovine urine.

[0087] The results in Table 5 show that, even with the addition of colchicine to bovine urine, the antibacterial activity of leukocytes was suppressed, and a sufficient number of bacteria (E. coli) contained in the bovine urine could be cultured.

[0088] The results from Examples 1-3 showed that the body fluid sampling container according to this embodiment can suppress the antibacterial activity of leukocytes in cow's milk, goat's milk, and cow's urine, and can culture a sufficient number of bacteria to identify the types of bacteria contained in each leukocyte-containing body fluid. Therefore, it was found that the body fluid sampling container according to this embodiment is not limited by the type of subject or leukocyte-containing body fluid, but can be widely used to identify types of bacteria. [Explanation of Symbols]

[0089] 1. Body fluid sampling container 2 containers 3. Non-bactericidal leukocyte function inhibitors 4 Filter paper 5 Body fluid containing white blood cells

Claims

1. A container for sampling body fluid containing leukocytes, in order to identify the types of bacteria contained in the body fluid taken from a subject, Based on the volume of leukocyte-containing body fluid, it contains 0.1 μg / mL to 1 μg / mL of colchicine or 0.2 mg / mL to 5 mg / mL of EDTA (ethylenediaminetetraacetic acid). A body fluid sampling container characterized in that the subject is a cow or a goat, and the leukocyte-containing body fluid is milk collected from a cow or goat suffering from mastitis.

2. The bodily fluid sampling container includes filter paper inside the container. The body fluid sampling container according to claim 1, characterized in that the filter paper contains colchicine or EDTA (ethylenediaminetetraacetic acid).

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